DEFINING THE ROLE OF CHROMATIN ARCHITECTURE IN CELL FATE INHERITANCE Proposal Summary The loss or destabilization of cell identity is central to a great spectrum of human disorders, including degenerative diseases and malignancies. A given cell identity is determined by specific gene expression programs, unique patterns of DNA and histone marks and ? a feature that only recently has become appreciated - characteristic three-dimensional (3D) genomic organization. During cell division most of these molecular features are temporarily erased due to extensive chromatin condensation, which poises a significant challenge to the continuity of cell memory. How cell identity is reestablished after mitosis constitutes a fundamental and -yet- unanswered question in molecular and cell biology. Here, we propose that a limited set of cell-type specific transcription factors together with architectural cofactors remain bound on selected sites of mitotic chromatin to ensure the faithful topological reorganization of the genome after cell division. The re-emerging chromatin architecture provides the necessary scaffold for successful reestablishment of the cell-defining molecular and functional characteristics. Employing a novel genetic approach that we designed in rapidly dividing mammalian stem cells, we will screen for transcription factors and architectural cofactors that are instrumental during the mitotic window to ensure faithful propagation of cell identity. Combining next generation sequencing approaches with single cell imaging in synchronized cells we expect to delineate for the first time the order of molecular events that instruct the successful reestablishment of cell identity and determine the role of chromatin organization. Finally, we will directly test to what extent temporal destabilization of selected architectural factors may enable manipulation of cell fate towards a desired direction. The successful completion of our goals may offer a solution to the longstanding puzzle of cell fate heritability after mitosis and point towards unconventional ways to alter cell identity with potentially major impact for regenerative medicine and cancer therapy.